Animal model for diabetic complications

ABSTRACT

The invention relates to the surprising find that low density lipoprotein receptor-deficient mice (LDLr −/− ) mice when fed with high energy diets produce controllable and consistent diabetic complications, especially renal damage, similar to the human pathophysiology and biological response. The invention thus comprises a method for discovering a preventive or therapeutic regimen for the prevention or treatment of diabetic micro- or macrovascular complications, comprising the steps of:
         a. feeding LDLr −/−  mice, which have not been treated with streptozotocin, with a high energy diet;   b. before, during and/or after this diet treating the mice with the preventive or therapeutic regimen;   c. checking whether any change in the micro- or macrovascular system of the animal occurs.       

     Specifically in such a method renal damage is assessed. Also use of said mice fed with a high energy diet for studying the diabetic micro- and macrovascular complications is part of the invention.

FIELD OF THE INVENTION

The invention relates to the field of diabetes, more particularlycomplications caused by diabetes or diabetes-like phenomena, likeinsulin resistance, metabolic syndrome and such, especially diabeticcomplications such as micro- and macrovascular complications,retinopathy, hepatopathy and diabetic nephropathy. More particularly,the invention relates to an animal model for such complications, inparticular diabetic nephropathy and methods for finding therapies forthe prevention of diabetes induced tissue damage and particularly renaldamage.

BACKGROUND

Nephropathy is a complication which is often seen in diabetes, as wellwith diabetes type 1, diabetes type 2, pre-diabetes, gestational asdrug-induced diabetes. There are wide differences in estimates of howmany people with diabetes will progress to having diabetic kidneydisease—from 6 to 27 percent of people with Type 1 diabetes, to 25 to 50percent of Type 2. Diabetic disease is characterized by high levels ofblood glucose which is associated with tissue damage. Small vascular andcapillary structures are very sensitive to diabetes-induced injury andorgans such as the kidney are frequently affected. By endothelial andmicrovascular damage the kidneys start to become ‘leaky’ which is firstdiagnosed on basis of increased protein levels in the urine(microalbuminuria). If no intervention follows, this develops in an evenhigher level of proteins in the urine (macroalbuminuria), glucosuria andfinally in end-stage renal disease (ESRD) which necessitates dialysis orkidney transplantation.

Diabetic nephropathy is the major cause of ESRD worldwide, and itsincidence has increased by more than 50% in the past ten years (USRDS2008 Annual Data Report: Atlas of Chronic Kidney Disease and End-StageRenal Disease in the Unites States, NIH).

Similar micro- and macrovascular complications that play a role in renaldamage also can result in other forms of complications, such asretinopathy. Is is likely that micro- and macrovascular dysfunction alsoplay a role in the development other diabetes-associated pathologiessuch as neuropathy, hepatopathy, hepatosteatosis, adipose inflammation(e.g. in visceral adiposity) and pancreas dysfunction.

Lack of reliable, translational animal models that sufficiently mimicthe multifactorial human pathofysiology have prevented optimaldevelopment of insight in the disease mechanism and candidatetherapy/drug development. Therefore, much effort has been devoted todevelop animal models for this specific complication in diabetes (see:Brosius, F. C. et al., 2009, J. Am. Soc. Nephrol. 20:2503-2512). Somemouse models have been described in this overview, but it is consideredthat there is still need for more, and/or more comprehensive animalmodels. Some of the drawbacks of the present models are: i) thatdiabetic complications develop heterogeneously with great variationsbetween animals (despite comparable genetic make-up and age) and ii)that the severity of diabetic complications, especiallymicroalbuminuria, does not progress over time as it is the case inhumans.

SUMMARY OF THE INVENTION

The inventors now have surprisingly found that low density lipoproteinreceptor-deficient mice (LDLr^(−/−)) mice when fed with high energydiets produce controllable, progressively developing and consistentdiabetic complications, especially renal damage, similar to the humanpathophysiology and biological response.

The invention thus comprises a method for discovering a preventive ortherapeutic regimen for the prevention or treatment of diabetic micro-or macrovascular complications, comprising the steps of:

-   -   a. feeding LDLr^(−/−) mice, which have not been treated with        streptozotocin, with a high energy diet;    -   b. before, during and/or after this diet treating the mice with        the preventive or therapeutic regimen;    -   c. checking whether any change in the micro- or macrovascular        system of the animal occurs.        More specifically, the invention relates to a method for        discovering a preventive or therapeutic regimen for the        prevention or treatment of diabetic nephropathy (and        microalbuminuria), comprising the steps of:    -   a. feeding LDLr^(−/−) mice, which have not been treated with        streptozotocin, with a high energy diet;    -   b. before, during and/or after this diet treating the mice with        the preventive or therapeutic regimen;    -   c. checking whether any change in the protein content of the        urine of said mice occurs and or whether any histological or        immunohistological change in the kidneys of said mice occurs.        Preferably in a method according to the invention, the        preventive or therapeutic regimen is administration of a        medicament or combination of medicaments, wherein the        medicaments in said combination may be administered        concomitantly or sequentially. Alternatively the preventive or        therapeutic regimen is a life style intervention or a        combination of a life style intervention and one or more        medicaments.        Also part of the invention is the use of LDLr^(−/−) mice for        study regarding the aetiology, progression, prevention, and        treatment of diabetic macro- and microvascular complications, in        which mice said complications have not been effected by        streptozotocin induced diabetes. More specifically, these        diabetic complications are selected from the group of diabetic        neuropathy, diabetic hepatosteatitis, impaired wound healing,        glucosuria and diabetic retinopathy, or combinations thereof.

LEGENDS TO THE FIGURES

FIG. 1: The increase in body weight is correlated to the concentrationof fat in the diet.

FIG. 2: HOMA values are dose-dependently increased by intake of fatthrough the diet.

FIG. 3: Dose dependent increase in urinary protein concentration(Y-axis) in relation to fat content in the diet. The protein content ofthe urine is generally expressed as the albumin to creatinine ratio,where the level of albumin is typically expressed in μg/ml while thelevel of creatinine is typically expressed in mg/ml.

FIG. 4: ALAT-values caused by high-energy diets.

FIG. 5: Body weight in LDLr^(−/−) mice fed with high energy diet afterpharmaceutical and life style interventions.

FIG. 6: Gonadal fat mass in LDLr^(−/−) mice fed with high energy dietafter pharmaceutical and life style interventions.

FIG. 7: Concentration of protein in urine in LDLr^(−/−) mice fed withhigh energy diet after pharmaceutical and life style interventions.

FIG. 8: Histology of the liver. Clearly shown are the lipid droplets andcollagen depositions.

DETAILED DESCRIPTION

In the following description and examples a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. Unless otherwise defined herein,all technical and scientific terms used have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

LDLr^(−/−) mice as first described by Ishibashi, S. et al. in 1994 (J.Clin. Invest. 93:1885-1893, 1994) are mice that do not or no longerexpress an active LDL receptor protein, preferably in which the genecoding for said LDL-receptor has been knocked out. They are commonlyused for research purposes in the field of lipid metabolism andatherosclerosis.

Protein content of the urine is expressed as the albumin/creatinineratio as is discussed in detail in the Examples.

Nephropathy or renal damage is the condition in which the function ofthe kidneys is deteriorated, which condition includes morphologicalmanifestations of renal tissue damage, microalbuminuria,macroalbuminuria, fatty degeneration, tissue necrosis, glucosuria,kidney fibrosis and end-stage renal disease (ESRD).

High energy diets are diets in which the lipid content is increased,especially in the form of fats or oils. Preferably animal fats, such aslard, beef tallow, butter, and ghee are used. These diets are typicaldiabetogenic diets and they also induce obesity. Some plant-derived fatsare also diabetogenic, for instance palm oil based diets. All the abovediabetogenic diets contain a high content of saturated fatty acids andmonosaturated fatty acids and relatively little polyunsaturated fattyacids (PUFAs). When diabetogenic diets are fed to susceptible animals,such as LDLr^(−/−) mice or apoE3 Leiden mice, these animals will develophuman-like features of the metabolic syndrome and/or diabetes.

LDLr^(−/−) mice have been used already for some years in the researchfor atherosclerosis using atherogenic diets. Atherogenic diets differfrom diabetogenic diets in several ways: they typically contain less fat(in w/w % as well as in En %) and they typically are supplemented withcholesterol at a concentration of 0.1% w/w and 1% w/w). Someinvestigators also add chocolate to the atherogenic diets to stimulatecholesterol uptake and to increase plasma cholesterol. A typicalatherogenic diet contains for instance 15% w/w cocoa and 1% w/w palm oiland 1% cholesterol as fat source (as reported in Kleemann et al. GenomeBiology, 2007). An overview of the most frequently used atherogenicdiets (clearly differing from diabetogenic diets) is also provided inZadelaar et al., ATVB, 2007). The LDLR−/− mice, being deficient for theLDL-receptor protein have been found to have severe elevated plasma LDLcholesterol levels when fed a atherogenic diet. and increased LDLcholesterol is associated with aortic atherosclerotic lesiondevelopement (Ishibashi, S. et al., 1994, J. Clin. Invest.93:1885-1893).

In these mice it has also been proven feasible to introduce a diabeteslike syndrome by treating them with a chemical substance that targetsthe pancreas, streptozocin (STZ), a method that has been applied inseveral animal models to introduce a diabetes type 1 like state bydamaging pancreatic cells by STZ (McEvoy, R. C. et al., 1984, J. Clin.Invest. 74:715-722). Further, it is known that STZ-induction of type 1diabetes in LDLR^(−/−) mice is associated with (spontaneousnephropathy(Hammad, S. M., 2003, Kidney Blood Press. Res. 26:351-361; Taneja, D.,et al. 2010, J. Lipid Res. 51:1464-1470).

The present inventors now surprisingly found that feeding LDLr^(−/−)mice with a high energy diet did not only increase plasma LDLcholesterol, triglyceride levels, plasma glucose levels, plasma sVCAM-1,plasma E-selectin, plasma SAA, plasma insulin levels and plasmaC-peptide levels (the latter clearly demonstrating intact pancreaticbeta cells) thereby enabling their uses as animal model for insulinresistance/metabolic syndrome and atherosclerosisbut they also foundthat these mice developed diabetic complications, in particular anephropathy, as revealed by microalbuminuria, which behaveddose-dependently in respect to the amount of fat ingested with the food.This nephropathy thus developed under conditions leading todiet-inducible type 2 diabetes and without an STZ induction, and thusnot only resembles the human aetiology of the disease, but also led tosimilar, micro- as well as macrovascular complications andpathophysiology. Notably, LDLR−/− mice fed diabetogenic diets exhibitelevated plasma insulin levels and elevated C-peptide levels at the sametime point when microalbuminuria is observed indicating that pancreaticbeta cells are still intact and functioning (C-peptide is co-secretedwith insulin from pancreatic beta-cells). This clearly demonstrates thatthe microalbuminuria of the present invention is unrelated to theSTZ-induced microalbuminuria which has been reported by others (seereferences above).

The high energy diet caused already notable symptoms of diabetic diseaseincluding microalbuminuria within 10-15 weeks after the start of highfat diet feeding, which means that the animal model of the currentinvention can be obtained relatively rapidly, and is thus suitable forpharmacological studies.

Importantly, diabetic complications in this model i) are diet-induciblein a dose-dependent way; ii) renal disease also progresses over time(increasing extent of microalbuminuria); and iii) the complications,especially the renal damage, are similar to the human pathophysiologyand biological responses. Lastly, the extent of renal disease isreversible and can be modulated by both dietary interventions (includinglifestyle) as well as with drugs.

The animal is very well suited to study the aetiology of diabeticnephropathy and the influences of external factors, drugs, lifestylechanges (such as change in diet and/or body exercise), aging, hormonalstatus, stress, inflammatory tone on the development of the nephropathy.Possible therapeutic interventions, not only for the prevention ofdiabetic nephropathy, but also for the treatment of this complicationcan easily be accommodated by using this animal model. The severity ofthe disease state can easily be monitored by determining the amount ofprotein in the urine (albuminuria).

Accordingly, the invention relates to a new method for discoveringpreventive and therapeutic strategies for the prevention or treatment ofdiabetic nephropathy comprising the steps of a) feeding LDLr^(−/−) micewith a high energy diet; b) before, during and/or after this diettreating the mice with the preventive or therapeutic regimen and c)checking whether any change in the protein content of the urine of saidmice occurs. It should be understood that the ‘preventive or therapeuticregimen’ mentioned above can be a medicament or a combination ofmedicaments, including neutraceuticals and biologicals (administeredconcomitantly or sequentially), but also a change in lifestyle, or acombination of both. A change in lifestyle may include a change toanother diet, or any further interference, such as a change in housingconditions (such as climatic conditions, day-night rhythm, socialenvironment (solitary or group housing condition)), a change in feedingpattern, alternating feeding, change in stress, change in hormonetreatment or a change in the level of physical exercise of the testanimals, with respect to the animals of the control treatment.

The new model also allows to study the effects of preventive andtherapeutic regimens on systemic and local inflammation.

Since the organs that are susceptible to diabetic complicationsincluding the kidney, the liver, the eye, the gut and the larger vesselsare surrounded by adipose tissue and since adipose tissue may contributeto the development of diabetic complications in these tissues, theapplication of this newly developed model also comprises testing ofpreventive and therapeutic regimens directed at improving the functionof liver (diabetic non-alcoholic steatosis, NASH and liver fibrosis),the function of the eyes (diabetic retinopathy), neuropathy inducedimpaired wound healing, the integrity and inflammatory state of the gutand the quantity and/or inflammatory status of adipose tissue itself. Ithas been shown in the experimental part of the present invention thatliver abnormalities show in the mice model of the invention.

The invention will be illustrated in the following Example(s), which isfor illustrative purpose and not deemed to be limiting the invention asclaimed.

EXAMPLES

Experimental Set-Up

Groups of male LDLr^(−/−) male mice (>12 weeks old) were fed a high fatdiet for 20 weeks and monitored over time. Groups were treated withincreasing concentrations of dietary fat as listed below (energypercentage from lard; En %) and a control group received chow.

-   -   1. chow control    -   2. 30 En % Lard diet    -   3. 45 En % Lard diet    -   4. 60 En % Lard diet

Body weight and food intake were measured over time. Tail blood samples(4 h fasting blood) and spot urine were taken at regular time points(time points are indicated in graphs below; last plasma sample was takenin week 18) and analyzed for glucose, insulin, HOMA (Homeostatic ModelAssessment), plasma lipids, systemic- and vascular inflammation as wellas ALAT (alanine transaminase). At the end of the experiment, organswere collected, weighed and stored for future analysis.

Results

The above experimental set-up allowed definition of the most optimalconcentration of lard in the diet to establish the hallmarks of humanprediabetes, insulin resistance and overt diabetes.

A dose dependent increase in body weight was observed with increasingamount of dietary fat (FIG. 1). This increase in body weight wasreflected by dose dependent increase in visceral- and subcutaneousadipose tissue. Both adipose depots have causatively been associatedwith the development cardiovascular and metabolic disease. By contrast,gonadal adipose tissue was comparably increased in the fat-treatedgroups. After 9 weeks of high fat treatment, the parameters indicativefor prediabetes were manifest.

For example, glucose and insulin were markedly elevated by the twohighest lard concentration and consequently also the HOMA valueindicating a state of insulin resistance (FIG. 2). Plasma glucose levelsin week 15 were 11.8 mM, 13.9 mM, 15.7 mM and 16.5 for group 1, 2, 3 and4, respectively. Plasma C-peptide levels (C-peptide is co-secreted withinsulin but has a greater plasma half-life) in week 12 were 2.0, 3.0,3.1 and 3.7 ng/mL for group 1, 2, 3 and 4, respectively. Plasmacholesterol and plasma triglycerides were markedly elevated by alldiets. More specifically, plasma cholesterol levels in week 18 were 8,19, 24, 26 mM and plasma triglyceride levels in week 18 were 1, 3, 4, 5mM for group 1, 2, 3, and 4, respectively. The increase in total plasmacholesterol was mainly confined to cholesterol in LDL particles (asrevealed by lipoprotein analysis, not shown). Systemic inflammation(quantified by serum amyloid A in plasma) was 2.5, 7.1, 8.3, 22.8 μg/mLfor group 1, 2, 3, and 4, respectively, and significantly increased withall three lard diets. Also vascular inflammation markers (VCAM andE-selectin) were significantly elevated indicating macro- andmicrovascular dysfunction, respectively (e.g. E-selectin in week 12: 79,89, 86 and 90 ng/mL for group 1, 2, 3, and 4, respectively).

In line with microvascular damage, a dose dependent increase inmicroalbuminurea was observed (FIG. 3) which is a hallmark ofnephropathy. Interestingly, the model exhibited a diet-inducible,homogenously developing microalbuminuria that developed gradually overtime and in a dose-dependent fashion depending on the amount of fat inthe diet. Finally, ALAT, a measure of liver function, was only slightlyincreased by 30% and 45% En lard, but was markedly increased by 60% Enlard (FIG. 4).

Example 2

Based on the findings of the above experiment, an intervention study wasdesigned. The middle dose (45 En % lard diet) was defined to be theoptimal diet to use for interventions with drugs since ALAT was notincreased Thereby putative adverse liver effects of drugs can still bedetected. Several anti-diabetic interventions (drugs and life-style)were tested according to the experimental set-up described above.

LDLr^(−/−) mice (12 weeks old) were fed 45 En % Lard diet for 9 weeks toestablish a prediabetic state (as described above and including visceraladiposity, hypertriglyceridemia, elevated glucose and insulin, chronicinflammation). After these 9 weeks, the interventions were started andcontinued until week 16. Drugs were mixed into lard diet andadministered orally via the feed. One group of mice was sacrifice at thestart of the intervention at week 9 for reference. Another control groupwas fed chow throughout the 16 weeks (serving as internal reference).The groups were as follows:

1. Chow 15 weeks: healthy control 2. Lard 15 weeks: diseased control{close oversize brace} controls 3. Lard 9 weeks: start interventioncontrol 4. Metformin (250 mg/kg) 5. Sulphonylurea (Glibenclamide 10mg/kg) 6. Rosiglitazone (10 mg/kg) 7. Pioglitazone (10 mg/kg) 8. DDP-IVinhibitor (Sitagliptin 20 mg/kg) 9. Fenofibrate (50 mg/kg) 10.Atovastatin (10 mg/kg) 11. LXR agonist (T0901317, 10 mg/kg) 12. Vioxx(34 mg/kg) 13. Salicylate (40 mg/kg) 14. Life style intervention (after9 weeks Lard switch to chow)Body weight and food intake were measured regularly and blood sampleswere taken at t=0, t=6, t=9, t=12, t=14 and t=15 weeks. Spot urine wascollected at t=0, t=8 and t=15 weeks. At the end of the study, the micewere sacrificed and the various organs were isolated and weighed andstored at −80 C and/or in formalin. Livers were processed for microarrayanalysis. More specifically, the same lobe of each liver was used forRNA extraction to prepare mRNA of microarray quality according to anestablished protocol (Kleemann et al., Genome Biology, 2007).

Results of the intervention study: Mice that were fed lard for 16 weeksbecame obese while mice which were kept on chow did not develop symptomsof prediabetes confirming that the disease phenotype is induced by thediet. The first data demonstrate that the different interventions havedifferential effects on global health parameters (body weight, tissueweights, fat distribution over the various depots etc) and onmicroalbuminuria which appears to be reversible and which can bemodulated with drugs. For instance, body weight (FIG. 5) was reduced byfenofibrate and LXR agonist, whereas body weight was slightly increasedby rosiglitazone. Total adipose tissue was unaffected by rosiglitazone,while the distribution of the fat across the various depots was markedlyaffected with this drug (subcutaneous fat was increased while gonadalfat mass (FIG. 6) was reduced). Gonadal fat was also lowered byfenofibrate, sulphonylurea, LXR agonist and salicylate.

Interestingly, differential effects of the drugs on microalbuminuia wereobserved. Microalbuminuria is a hallmark of diabetic nephropathy and wasdetermined by measuring the concentration of albumine and creatinine inspot urine collected at various time points during the experiment.Subsequently the albumine/creatinine ratio was calculated, viz. theamount of albumine expressed in μg per mg creatinine (FIG. 7). The drugsmetformin, fenofibrate, atorvastatin, glibenclamide, rosiglitazone,pioglitazone and salicylate significantly improved thealbumine/creatinine ratio compared to high fat treated control animalsthat were not treated with drugs. Since drugs were mixed into the highenergy diet, the data indicate that these drugs interfere withdiet-evoked diabetic nephropathy in a setting of type 2 diabetes.Notably, lifestyle intervention (switch to chow feeding) also reducedmicroalbuminuria to a level that was comparable to healthy controlanimals (FIG. 7) demonstrating that the disease phenotype is fullyreversible.

Example 3

Male LDLr^(−/−) mice (>12 weeks old) were fed a high fat diet (45% En %Lard diet) for 10 weeks, and for an additional 6 weeks high fat dietenriched with 1% (w/w) cholesterol.

Animals were sacrificed and livers were removed. Liver was weighed,fixed in formaldehyde and paraffin embedded. Tissue sections werestained for collagen using picrosiriusred staining (0.1% Sirius Red insaturated aqueous picric acid solution) and analyzed microscopically.

It was observed (FIG. 8) that the histology very much resembled thehistology seen in human steatosis/NASH (non-alcoholic liver steatohepatitis), as shown by Cohen, J. C. et al, Science 3332, 1519-1532, 24Jun. 2011. Clear deposition of lipid droplets in the cytoplasm ofhepatocytes, and significant collagen deposition (fibrosis) could beobserved (FIG. 8). This means that the specific diet treated mice of theinvention can also be used to assess the liver for steatosis likephenomena.

1-8. (canceled)
 9. A method for discovering a preventive or therapeuticregimen for the prevention or treatment of diet-induced liver steatosis,non alcoholic steato hepatitis (NASH) and liver fibrosis, comprising thesteps of: (a) feeding LDLr^(−/−) mice, which have not been treated withstreptozotocin with a high energy diet without supplemented cholesterol;(b) before, during or after this diet treating the mice with thepreventive or therapeutic regimen; and (c) checking whether any changein liver histology of said mice occurs.
 10. The method of claim 9,wherein the preventive or therapeutic regimen has been administration ofa medicament or combination of medicaments, wherein the medicaments insaid combination may be administered concomitantly or sequentially. 11.The method of claim 9, wherein the preventive or therapeutic regimen hasbeen a lifestyle intervention or a combination of a life styleintervention and one or more medicaments.
 12. The method of claim 9,wherein said preventive or therapeutic regimen is selected from: (a) achange of diet, (b) a change of housing conditions, (c) a change infeeding pattern, (d) alternating feeding, (e) a change in stress, (f) achange in hormone treatment, and/or (g) a change in the level ofphysical exercise.
 13. The method of claim 9, wherein the high energydiet without supplemented cholesterol comprises animal fats selectedfrom lard, beef tallow, butter and ghee.